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Series GSE5184

Query DataSets for GSE5184

Status

Public on Dec 10, 2007

Title

PGC1alpha overexpression in human white adipose cells

Organism

Homo sapiens

Experiment type

Expression profiling by array

Summary

Subcutaneous abdominal adipose tissue was obtained from female subjects undergoing plastic surgery in agreement with French laws on biomedical research. Stromal cells prepared from WAT were cultured for 13 days in a chemically defined medium. At day 13, 60–80% of cells were differentiated into lipid droplet-containing adipocytes. The cells were infected at a m.o.i. of 200 for 6 h. The day after infection, cells were treated with the following drugs at 1 µM unless otherwise indicated: rosiglitazone (BRL49653, Smith Kline and French, Harlow, UK) and 9-cis-RA (Sigma). Cells were harvested after 48 h of treatment. PPAR agonists and 9-cis-RA were prepared in dimethylsulfoxide, and the remaining compounds were diluted in medium. Dimethylsulfoxide or medium was added to control cells) to obtain the same concentration as in compound-treated conditions.

Human subcutaneous abdominal adipose tissue was obtained from female subjects undergoing plastic surgery in agreement with French laws on biomedical research. In brief, on 100 ml of tissue, all visible fibrous material and blood vessels were discarded. Adipose tissue was subjected to collagenase digestion. After centrifugations, stromal cells were suspended in Dulbecco's modified Eagle's medium (DMEM)/Ham's F-12 medium supplemented with 10% fetal calf serum (FCS) and antibiotics. Cells were seeded at 30,000 cells/cm2. After 20h incubation, the medium was changed to a chemically defined differentiation medium, consisting of DMEM/Ham's F-12 supplemented with biotin (33 5mol/l), panthotenate (17 5mol/l), insulin (66 nmol/l), triiodothyronine (1 nmol/l), cortisol (100 nmol/l), transferrin (100 ng/ml), (50 5g/ml) gentamicin and (0.2u/ml) penicillin/streptomycin and ciglitazone (1 5g/ml) the first 3 days. Cells then received the same medium without ciglitazone which was changed every 3rd day. At day 13, 60 80% of cells were differentiated into lipid droplet-containing adipocytes.
The full-length human PGC-1alpha; cDNA was cloned into the pAdEasy parent plasmid. Recombination between the pAdEasy and pAdTrack vectors and production of the PGC-1alpha; adenovirus was performed at the Laboratoire de Therapie Genique de Nantes. The virus contains, in tandem, the green fluorescent protein (GFP) gene and the PGC-1alpha; cDNA downstream of separate cytomegalovirus promoters. An adenovirus containing only the GFP gene was used as control. Viral titers were 1011 infectious particles per ml.
For experiments with agonist treatments, cells were infected with PGC1alpha; adenovirus at a m.o.i. of 200 for 6 h. The day after infection, cells were treated as indicated in text and figure legends with the following drugs at 1 5M unless otherwise indicated: rosiglitazone (BRL49653, Smith Kline and French) and 9-cis-retinoic acid (Sigma). Cells were harvested after 48 h of treatment. PPAR agonists and 9-cis-RA were prepared in dimethylsulfoxide then diluted in medium. A similar dimethylsulfoxide was added to control cells as in drug-treated cells.
Total RNA was extracted using the Qiagen RNeasy kit (Qiagen, Courtaboeuf, France, RNA concentration and integrity were assessed using Agilent 2100 Bioanalyzer (Agilent Technologies, Massy France). In order to compare microarray experiments, we used a common reference pool which was generated by mixing equal amounts of total RNA extracted from control samples. One 5g of total RNA from each total RNA sample preparation was amplified by MessageAmp RNA Kit (Ambion, Huntington, Cambridgeshire, UK) and amplified RNA quality checked using Agilent 2100 Bioanalyser (Agilent Technologies, Massy France). 35g of amplified RNA was labeled with cyanin dyes (Cy) using the CyScribe First-Strand cDNA labeling kit (Amersham Biosciences, Orsay, France). Amplified RNA from the reference pool was labelled with Cy3 and those from the testing samples were labelled with Cy5. The labelled cDNA mixtures were hybridized according to the protocol described at http://cmgm.stanford.edu/pbrown/protocols/index.After scanning the arrays, the images were analyzed using Genepix Pro 5.1 software and entered into SMD.
Keywords: treatment comparison

Overall design

4 types of tratment were tested:
control (vehicle) cells
cells overexpressiong PGC1 alpha
cells overexpressiong PGC1 alpha and treated with BRL 49653
cells treated with BRL 49653

An amplification and labeling experiment design type is where different amplification and/or labeling methods are compared.

Contributor(s)

Mazzucotelli A, Viguerie N, Lepin E, Langin D, Clément K

Citation(s)

17646210

Submission date

Jun 28, 2006

Last update date

Mar 16, 2012

Contact name

Karine Clement

E-mail(s)

karine.clement@htd.ap-hop-paris.fr